ORIGINAL PAPER

Efficient sweet pepper transformation mediated by the BABYBOOM transcription factor

Iris Heidmann • Brenda de Lange • Joep Lambalk •

Gerco C. Angenent • Kim Boutilier

Received: 23 November 2010 / Revised: 18 January 2011 / Accepted: 18 January 2011 / Published online: 9 February 2011

� The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract Pepper (Capsicum L.) is a nutritionally and

economically important crop that is cultivated throughout

the world as a vegetable, condiment, and food additive.

Genetic transformation using Agrobacterium tumefaciens

(agrobacterium) is a powerful biotechnology tool that

could be used in pepper to develop community-based

functional genomics resources and to introduce important

agronomic traits. However, pepper is considered to be

highly recalcitrant for agrobacterium-mediated transfor-

mation, and current transformation protocols are either

inefficient, cumbersome or highly genotype dependent.

The main bottleneck in pepper transformation is the

inability to generate cells that are competent for both

regeneration and transformation. Here, we report that

ectopic expression of the Brassica napus BABY BOOM

AP2/ERF transcription factor overcomes this bottleneck

and can be used to efficiently regenerate transgenic plants

from otherwise recalcitrant sweet pepper (C. annuum)

varieties. Transient activation of BABY BOOM in the

progeny plants induced prolific cell regeneration and was

used to produce a large number of somatic embryos that

could be converted readily to seedlings. The data highlight

the utility of combining biotechnology and classical plant

tissue culture approaches to develop an efficient transfor-

mation and regeneration system for a highly recalcitrant

vegetable crop.

Keywords Sweet pepper (Capsicum annuum) �Transformation � Agrobacterium � BABY BOOM �Somatic embryogenesis � Regeneration

Abbreviations

TDZ Thidiazuron

BBM BABY BOOM

CCM Co-cultivation medium

EM Elongation medium

GR Rat glucocorticoid receptor ligand binding domain

GUS b-Glucuronidase

MS Murashige and Skoog medium

PRM Pre-rooting medium

RM Rooting medium

SE Somatic embryo

SLS Shoot-like structures

Introduction

The genus Solanaceae comprises some of the most eco-

nomically important vegetable species, including potato

(Solanum tuberosum), tomato (Solanum lycopersicon),

eggplant (Solanum melongena), and pepper (Capsicum

spp.). More than 40 species belong to the genus Capsicum.

Five pepper species, C. annuum, C. frutescens, C. baccatum,

C. chinense, and C. pubescens, are valuable crops plants that

Communicated by L. Jouanin.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-011-1018-x) contains supplementarymaterial, which is available to authorized users.

I. Heidmann � B. de Lange � J. Lambalk

Enza Zaden Research and Development B.V, P.O. Box 7,

1600 AA Enkhuizen, The Netherlands

G. C. Angenent � K. Boutilier (&)

Plant Research International, Wageningen University

and Research Centre, P.O. Box 619, 6700 AP Wageningen,

The Netherlands

e-mail: kim.boutilier@wur.nl

123

Plant Cell Rep (2011) 30:1107–1115

DOI 10.1007/s00299-011-1018-x

are cultivated and consumed throughout the world, with

C. annuum being the most widely cultivated species. Pepper

is second only to tomato in terms of vegetable production in

developed countries, and its breeding and production, as with

other major crops, is constantly challenged by numerous

pests, diseases, and abiotic stresses (Djian-Caporalino et al.

2007). Trait development in the genus Capsicum is ham-

pered by interspecific crossing barriers, as well as by the

general lack of an efficient regeneration system, which is a

prerequisite for the introduction of new traits by genetic

transformation. The major bottlenecks in Capsicum regen-

eration are the general low frequency of shoot formation and

the development of malformed shoot buds and shoots (var-

iously referred to in the literature as rosette shoots, leafy

shoots or blind leaves) that fail to elongate, most likely due to

the absence of a shoot apical meristem (Dabauza and Pena

2003; Engler et al. 1993; Kothari et al. 2010; Liu et al. 1990;

Mihalka et al. 2003; Wolf et al. 2001). In general, the chili

(hot) pepper types are much more responsive for in vitro

regeneration than the sweet pepper types (Dabauza and Pena

2001; Engler et al. 1993; Khan et al. 2006; Lopez-Puc et al.

2006; Ochoa-Alejo and Ramirez-Malagon 2001; Solıs-

Ramos et al. 2010; Zapata-Castillo et al. 2007) although a

strong genotype dependency has been observed in both.

Agrobacterium tumefaciens (agrobacterium)-mediated

transformation of C. annuum has been described in the

literature, but in most reports only a few transgenic lines

were obtained, and/or the transformation efficiency and

heritability of the transgene were not reported (Dabauza

and Pena 2003; Manoharan et al. 1998; Mihalka et al.

2003; Shivegowda et al. 2002; Zhu et al. 1996). Repro-

ducible agrobacterium-mediated transformation is cur-

rently limited to a few responsive chili pepper genotypes

(Ko et al. 2007; Lee et al. 2004) and one sweet-mini pepper

genotype (Engler et al. 1993; Harpster et al. 2002). The

difficulties associated with pepper transformation have

been attributed to its low regeneration capacity, and the

poor overlap between the tissues that are competent for

agrobacterium infection and those that are competent for

regeneration (Wolf et al. 2001). This is a general phe-

nomenon that has been observed in plants that are recal-

citrant for transformation (Potrykus 1991). A system that

supports transformation and regeneration of the same tis-

sues could therefore provide the basis for an efficient

pepper transformation protocol.

A number of genes encoding transcription factors, cell

cycle proteins, and components of hormone biosynthesis

and signaling pathways have been shown to enhance plant

regeneration responses when mutated or ectopically

expressed (Banno et al. 2001; Catterou et al. 2002; Lotan

et al. 1998; Riou-Khamlichi et al. 1999; Zuo et al. 2002).

One of these genes, BABY BOOM (BBM), encodes an AP2/

ERF transcription factor that induces regeneration under

culture conditions that normally do not support regenera-

tion in wild-type plants. Ectopic expression of Brassica

napus BBM (BnBBM) genes in B. napus and the related

crucifer arabidopsis (Arabidopsis thaliana) induces spon-

taneous somatic embryogenesis and organogenesis from

seedlings grown on hormone-free basal medium (Boutilier

et al. 2002). In tobacco, ectopic BBM expression is suffi-

cient to induce adventitious shoot and root regeneration on

basal medium, but exogenous cytokinin is required for

somatic embryo (SE) formation (Srinivasan et al. 2007).

Ectopic BBM expression has also been used to generate

transgenic Chinese white poplar (Populus tomentosa Carr.)

plants (Deng et al. 2009). Poplar callus transformed with a

B. rapa BBM gene developed SEs that could be converted

into plantlets, while untransformed callus failed to regen-

erate. The system was combined with heat shock-inducible

FRT/FLP-mediated excision of the transgene to produce

marker-free lines.

Transformation strategies based on standard tissue cul-

ture approaches have not led to efficient pepper transfor-

mation protocols. We therefore examined whether the

positive influence of BBM expression on regeneration that

is observed in other plant species could be transferred to

pepper. Here, we describe the efficient regeneration of

large numbers of fertile transgenic plants of two C. annuum

sweet pepper varieties by combining a classical tissue

culture approach with transient activation of a BnBBM

protein.

Materials and methods

Explant pre-culture

Surface sterilized seeds of the F1 hybrids Fiesta, Ferrari,

and Spirit (Enza Zaden, The Netherlands) were sown on

full strength MS medium (Murashige and Skoog 1962)

with 2% (w/v) sucrose (pH 5.8, MS20) solidified with 0.8%

(w/v) Microagar. Ten-day-old petiole-free cotyledons were

cut twice, transverse to the mid-rib, to generate three

explants, which were then pre-cultured on solid (0.7%

Microagar) co-cultivation medium (CCM) supplemented

with 40 mg/l acetosyringone (Acros Organics) for 1–2 days

under dim light conditions (1,500 lux) at 23�C. CCM is a

modified R medium (Sibi et al. 1979) supplemented with

1.6% (w/v) glucose, 2 mg/l zeatin riboside and 0.1 mg/l

indole-3-acetic acid to promote shoot regeneration.

Agrobacterium and vectors

Agrobacterium strain GV3101 carrying the pMP90 Ti

plasmid was used in all experiments. Agrobacterium con-

taining the 35S::BnBBM:GR (Srinivasan et al. 2007) and

1108 Plant Cell Rep (2011) 30:1107–1115

123

35S::GUS binary vectors were grown with the appropriate

antibiotics in 100 ml YEB medium at 28�C. Prior to

transformation the agrobacterium suspension was diluted to

OD660 0.3–0.4 with liquid CCM supplemented with freshly

prepared 40 mg/l acetosyringone.

Transformation and regeneration

The diluted agrobacterium culture was added to the pre-

cultured cotyledon explants and incubated at room tem-

perature for 30–60 min. Explants were blotted dry and

further co-cultured on CCM supplemented with 40 mg/l

acetosyringone for 2–3 days under dim light conditions

(1,500 lux) at 23�C before transfer to selection medium

consisting of CCM supplemented with 1 mg/l thidiazuron

(TDZ), 100 mg/l kanamycin sulfate and 500 mg/l cefo-

taxime. Explants were transferred to full light conditions

(3,000 lux) on a 16 h/8 h day/night cycle at 23�C for

2 months. The medium was refreshed after 4 weeks.

Explants with emerging shoots or shoot-like structures

(SLS) were transferred for 4 weeks to elongation medium

(EM) consisting of MS macro- and microsalts (Murashige

and Skoog 1962), B5 vitamins (Gamborg et al. 1968), 1.6%

(w/v) glucose, 1 mg/l inositol, 20 mg/l adenine sulfate,

200 mg/l casein hydrolysate, 10 mg/l gibberellic acid 3,

4 mg/l benzylaminopurine and 30 lM silverthiosulfate.

Elongated shoots were transferred to pre-rooting medium

(PRM), comprising MS20 medium supplemented with

30 mg/l glutathione, 60 mg/l kanamycin sulfate, and

300 mg/l cefotaxime. The shoots were transferred after

1 month to rooting medium (RM; Rugini 1984) supple-

mented with 2% (w/v) sucrose, 50 mg/l kanamycin. Rooted

shoots were transferred into the greenhouse for seed set.

All media used in experiments involving the 35S::BnBBM:

GR construct were supplemented with 10 lM dexametha-

sone (DEX; Sigma) up to the point where shoots were

transferred to EM, after which DEX-free media was

used. Except where noted, all tissue culture chemicals

were supplied by Duchefa Biochemicals (Haarlem, The

Netherlands).

b-Glucuronidase (GUS) staining

Histochemical GUS staining (Jefferson 1987) of 35S::GUS

explants was performed after 3 weeks on selection

medium.

Evaluation of stable transgenic lines

of 35S::BnBBM:GR

Surface sterilized seeds were sown on MS20 medium

supplemented with either 10 lM DEX, 1 mg/l TDZ, or

10 lM DEX plus 1 mg/l TDZ. The response of the seed-

lings was evaluated 24 days after sowing.

Root, hypocotyl, and cotyledon explants from 10-day-

old seedlings and leaf explants from 4-week-old plantlets

were obtained from donor material grown on MS20

without any supplements. Explants were placed on MS20

or MS20 supplemented with 10 lM DEX, either alone or

in combination with 1 mg/l TDZ, benzylaminopurine or

zeatin riboside. The response of the explants was eval-

uated after 2 weeks. Conversion of SEs into plantlets

was assessed by placing embryos onto RM. Conversion

into plantlets was evaluated 4 weeks after transfer to

RM.

Results

BABY BOOM-mediated regeneration

We examined the utility of a BnBBM gene as a tool to

enhance regeneration during agrobacterium-mediated

transformation of C. annuum sweet, blocky pepper types.

Ectopic BBM expression induces pleiotropic phenotypes

such as adventitious growth and sterility that are likely to

interfere with the regeneration process and subsequent

growth of transgenic plants (Boutilier et al. 2002). To avoid

generating plants with detrimental phenotypes, we

expressed a fusion between the BnBBM protein and the

ligand binding domain of the rat glucocorticoid receptor

(BBM:GR), which sequesters the BBM transcription factor

in the cytoplasm in the absence of glucocorticoid steroid,

e.g., DEX (Passarinho et al. 2008; Srinivasan et al. 2007).

Explants were co-cultivated with agrobacterium carrying

either the 35S::BnBBM:GR construct or a control 35S::GUS

construct carrying the scorable GUS marker. Both con-

structs confer kanamycin resistance via the nptII selection

marker. Explants were co-cultivated with agrobacterium on

CCM and then transferred to CCM medium containing

kanamycin until shoots appeared, at which point the shoots

were transferred to EM. Elongated shoots were then

transferred to PRM and subsequently to RM when rooting

did not occur already on PRM. Approximately 5,600

explants from two cultivars were used in the transformation

experiments with the 35S::GUS plasmid, and approxi-

mately 6,400 explants from three cultivars in the trans-

formation experiments with the 35S::BnBBM:GR plasmid

(Table 1).

Cotyledon explants that were co-cultivated with agro-

bacterium containing the 35S::GUS construct behaved as

previously described for poorly regenerating or non-

transformable genotypes (Lee et al. 2004; Liu et al. 1990).

The explants increased in size about twofold during the

first 3 weeks on selection medium. Small calli became

Plant Cell Rep (2011) 30:1107–1115 1109

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visible at the cut edge of the explants during the fol-

lowing 2–4 weeks, accompanied by a few dense rosette-

forming SLS (Fig. 1a, b). SLS transferred to EM failed to

elongate and therefore did not root when transferred to

rooting medium (RM). In vitro grafting of the SLS onto a

wild-type rootstock did not promote further shoot devel-

opment or elongation, suggesting that the SLS lacked a

functional apical meristem. In a separate experiment,

histochemical staining of 3-week-old explants (n = 225)

that formed both SLS and callus showed that 6% of the

explants exhibited GUS activity and that the GUS activity

was restricted to the developing callus (Fig. 1c). This

observation supports the hypothesis of Wolf et al. (2001)

that under these conditions, pepper cells that are suscep-

tible for agrobacterium transfection lack regeneration

capacity and vice versa.

In contrast to the control experiments with the 35S::GUS

construct, co-cultivation of sweet pepper cotyledon

explants with agrobacterium carrying the 35S::BnBBM:GR

construct allowed us to generate numerous transgenic

Table 1 Regeneration response and transformation efficiency of sweet pepper varieties

Construct/cultivar No. of

explants

No. of

experiments

Explants with SLS

(% of total explants used)

Explants with

elongated shoots

No. transgenic

shootsaTransformation

efficiencyb

35S::GUS

Fiesta 5,150 24 64 (1.2) 0

Spirit 475 5 0 (0) 0

Total 5,625 29 64 0 0 0

35S::BnBBM:GR

Fiesta 4,448 25 798 (17.9) 26 78 0.6

Ferrari 805 6 29 (3.6) 9 20 1.1

Spirit 1,179 7 67 (5.7) 0 0

Total 6,432 38 894 35 98

a Individual explants produce multiple shootsb Transformation efficiency = (no. of explants with transgenic shoots/total no. of explants) 9 100%

Fig. 1 Regeneration response

of sweet pepper explants.

Response of sweet pepper

‘Fiesta’ explants to co-

cultivation with agrobacterium

carrying either the 35S:GUSconstruct (a–c) or the

35S::BnBBM:GR construct

(d–f). a Callus formation

(arrow) and shoot-like

structures (SLS; asterisk) after

3 weeks of culture; b leaf-like

structures developing at the

wounded edge of a cotyledon

after 4 weeks of culture;

c explant with callus (arrow)

and SLS (asterisk)

histochemically stained for

GUS activity; d SLS, 4 weeks

after treatment; e somatic

embryo formation (arrows) on a

newly emerged leaf, after

6 weeks of culture; f elongating

SLS (asterisk) after 4 weeks on

elongation medium

1110 Plant Cell Rep (2011) 30:1107–1115

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shoots that stably transmitted the transgene to the next

generation. The transformation experiments with the

35S::BnBBM:GR plasmid were carried out as described

above, except that 10 lM DEX was included in the

selection medium to localize the BBM:GR protein to the

nucleus. Explants co-cultivated with the 35S::BnBBM:GR

agrobacterium behaved essentially the same as in the

35S::GUS experiments during the first 7 weeks of culture,

except that they produced more SLS in each experiment

compared to the 35S::GUS control (Table 1). Unlike the

control experiments with 35S::GUS, the SLS that formed

after co-cultivation with the 35S::BBM:GR agrobacterium

elongated and proliferated within 4 weeks after transfer to

EM (Fig. 1d). SEs developed occasionally on the leaves of

elongated shoots and/or SLS that remained in contact with

the medium (Fig. 1e), and in turn produced elongated

shoots. Plantlet formation from elongated shoots was

enhanced after transfer to PRM (Fig. 1f). Shoots that did

not root on PRM formed roots within 2 weeks after transfer

to RM. An overview of the workflow for our sweet pepper

transformation protocol is shown in Table 2.

In total, 98 rooted shoots regenerated from 35 inde-

pendent SLS were transferred to the greenhouse. PCR on

T0 plants and germination of T1 seed on kanamycin-con-

taining medium suggested that all 98 plants were trans-

genic (data not shown and Supplementary Table 1). A

more detailed PCR analysis of 65 T1 lines derived from 31

SLS (Supplementary Fig. 1) showed that the 35S::BBM

fragment (located close to the right T-DNA border) could

be amplified in all 65 lines, while the nos::nptII fragment

(located close to the left border) could only be amplified in

62/65 lines (Supplementary Fig. 1). The three lines lacking

the nos::nptII fragment showed kanamycin resistance,

suggesting that the selection marker was truncated during

T-DNA integration but is still functional.

Regeneration and transformation efficiency

The regeneration response of the three tested varieties was

greatly enhanced in the experiments with the

35S::BnBBM:GR plasmid (Table 1) although genotypic

differences were observed among the varieties with respect

to the different steps in the regeneration protocol. In all

cases, co-cultivation with the 35S::BnBBM:GR plasmid

was able to relieve one or more bottlenecks in the regen-

eration/transformation procedure for each of the varieties

tested. Transgenic plantlets were generated for two of the

three varieties, whereas no transgenics were obtained from

any variety in the control experiments. Numerous elon-

gated shoots could be produced from a single SLS. How-

ever, as multiple shoots may arise from a single

transformation event, we only used a single elongated

shoot per SLS to calculate the transformation efficiency

(Table 1). Based on this criterion, we obtained average

transformation efficiencies of 0.6 and 1.1% for the two

varieties although the transformation efficiency can be

much higher in individual experiments (up to 3.8%, data

not shown). Different segregation patterns were often

observed among the progeny of the multiple shoots derived

from a given SLS (Supplementary Table 1), suggesting

that multiple independent transgenic plants can be regen-

erated from a single explant. TAIL-PCR was performed on

65 T1 lines derived from 31 SLS to determine whether

multiple lines from a single SLS correspond to clonal or

independent transgenic events. The TAIL-PCRs were not

successful for every set of SLS, but we were able to

identify five SLS in which the corresponding lines with

more than one TAIL PCR fragment. An example is shown

in Supplementary Fig. 2. Together, the data suggest that

the actual transformation efficiency is higher than calcu-

lated above.

Stable 35S::BnBBM:GR transformants are highly

regenerative

Two single locus, homozygous 35S::BnBBM:GR lines were

selected for further phenotypic analysis. Seeds were sown

on either MS20, MS20 supplemented with either DEX or

TDZ or MS20 supplemented with both DEX and TDZ.

Seedlings of the two 35S::BnBBM:GR lines were indistin-

guishable from the wild type when grown on MS20

(Fig. 2a). 35S::BnBBM:GR seedlings grown on DEX-con-

taining medium were severely delayed in their initial

growth as compared to wild-type plants growing on the

same medium. Seedlings from one of the two lines ger-

minated, but failed to develop further. Seedlings of both

Table 2 Workflow for 35S::BnBBM:GR-mediated sweet pepper

transformation

Step Days

(n)

Medium

Growth of donor material 10 MS20

Explant pre-culture 1–2 CCM ? ZR ? IAA

Co-cultivation of explants 3–4 CCM ? ZR ? IAA ? ACS

Shoot regeneration on

selective medium

2 9 30 CCM ? TDZ ? DEX

Shoot elongation 30 EM

Pre-rooting of elongated

shoots

30 PRM

Rooting of elongated shoots 14 RM

Total 150

CCM co-cultivation medium, MS20 Murashige and Skoog (1962)

with 2% (w/v) sucrose, ZR zeatin riboside, IAA indole-3-acetic acid,

ACS acetosyringone, TDZ thidiazuron, DEX dexamethasone, EMelongation medium, PRM pre-rooting medium, RM rooting medium

Plant Cell Rep (2011) 30:1107–1115 1111

123

lines showed a thickened root, a pronounced apical hook

(Fig. 2b) and were agravitropic. The cotyledons that

remained in contact with the medium eventually swelled

and formed irregular protruberances lacking a defined

structure. Wild-type and 35S::BnBBM:GR seedlings plated

on TDZ-containing medium developed as on the control

MS20 medium (Fig. 2c). Wild-type and 35S::BnBBM:GR

seedlings growing on medium supplemented with both

DEX and TDZ showed a combination of the phenotypes

observed in the presence of the individual compounds

(Fig. 2d). In addition, cotyledons of 35S::BnBBM:GR

seedlings that remained in contact with the medium

eventually developed into a callus mass and produced a

few SEs (data not shown).

Fig. 2 Regeneration response

of stable 35S::BBM:GR sweet

pepper lines. a–d 24-day-old

seedlings germinated on

medium with the indicated

supplements; e–h feather-cut

cotyledons of 10-day-old

seedlings incubated for 14 days

on medium with the indicated

supplements. Shoot-like

structures are indicated by an

arrow; i–l feather-cut leaves of

4-week-old plants incubated for

14 days on medium with the

indicated supplements;

m 35S::BBM:GR somatic

embryos; n immature wild-type

zygotic embryo; o somatic

embryo formation on wild-type

zygotic embryos. MS20Murashige and Skoog medium

with 2% (w/v) sucrose, TDZthidiazuron, DEXdexamethasone

1112 Plant Cell Rep (2011) 30:1107–1115

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The ability of 35S::BnBBM:GR explants to form SEs

prompted us to assess the regenerative capacity of dif-

ferent organs. Segments of roots and hypocotyls and

feather-cut cotyledons from 10-day-old seedlings, and

feather-cut leaves from 4-week-old homozygous

35S::BnBBM:GR plants were placed onto MS20 or MS20

supplemented with TDZ, DEX or both TDZ and DEX.

Root and hypocotyls segments formed callus but did not

regenerate under any of the conditions tested (data not

shown). Cotyledons and leaves cultured on MS20 formed

white callus at the wounded edges of the explant

(Fig. 2e, i). TDZ stimulated white callus production in

cotyledons and leaves, and also induced SLS formation

at the wounded edges of cotyledons (Fig. 2g, k). Cul-

turing cotyledons and leaves on medium with DEX

(Fig. 2f, j) or DEX and TDZ (Fig. 2h, l) induced prolific

SE formation. SE induction was greatly diminished when

uncut leaves and cotyledons were used as explants (data

not shown). Quantitative differences in SE production

were not observed when TDZ was replaced by the

cytokinins benzylaminopurine and zeatin riboside (data

not shown). SE induction was mainly observed on the

surface of the explant adjacent to the cut edge, and SEs

appeared to develop directly from the underlying tissue,

rather than indirectly through an intermediate callus

phase. SEs formed on DEX-containing medium generally

developed to the globular stage (Fig. 2f, j), while SEs

growing on medium containing TDZ (or other cytoki-

nins) and DEX progressed further to the heart-shaped

stage and beyond, in which the cotyledons are visible

(Fig. 2h, l). SEs from 35S::BBM:GR explants induced on

TDZ (or other cytokinins) and DEX have a clear bipolar

structure (Fig. 2m), and are more similar to wild-type

zygotic embryos (Fig. 2n) than to standard SEs derived

from immature wild-type zygotic embryos (Fig. 2o).

35S::BBM:GR SEs could be converted into plantlets by

plating them on RM. While individual cytokinins did not

affect SE production quantitatively, they did influence

the ability of SEs to convert into plantlets. The highest

conversion rate (50%) was obtained with embryos that

were induced in the presence of benzylaminopurine

(Supplementary Table 2).

Discussion

Pepper is a major crop that is grown world-wide and whose

production is threatened by various biotic and abiotic

stresses. Resistances can often be found in wild relatives,

but there are often strong breeding incompatibilities

between Capsicum species that are not easy to circumvent

(Jae et al. 2006; Onus and Pickersgill 2004). Traits from

incompatible wild relatives could be introduced into

cultivated peppers via genetic transformation; however,

peppers, especially the sweet genotypes, are highly recal-

citrant for transformation.

Here, we describe a reliable and efficient transformation

protocol for sweet pepper genotypes that takes advantage of

the enhanced regeneration response conferred by the BBM

AP2/ERF transcription factor. The protocol is straightfor-

ward in that additional measures such as grafting SLS onto a

rootstock (Mihalka et al. 2003), a long phase of shoot elon-

gation (‘‘normalization’’) (Engler et al. 1993) or the addition

of auxin to enhance rooting (Engler et al. 1993; Khan et al.

2006) were not required. Direct comparison of our sweet

pepper transformation efficiencies with published protocols

is difficult as in practice there are no routine, reliable and

reproducible protocols that are applicable to more than one

genotype. The comparison is further complicated by the lack

of information on the transformation efficiency (Harpster

et al. 2002; Zhu et al. 1996) or the heritability of the transgene

(Dabauza and Pena 2003; Engler et al. 1993; Manoharan

et al. 1998; Mihalka et al. 2003; Shivegowda et al. 2002). Our

transformation efficiencies of 0.6 and 1% obtained with two

recalcitrant sweet pepper genotypes are higher on average

than the 0.03–0.6% reported for the most responsive

C. annuum. chili pepper genotypes (Ko et al. 2007; Lee et al.

2004; Manoharan et al. 1998).

This and previous studies (Wolf et al. 2001) suggest that

the few regenerating structures that are obtained in stan-

dard pepper transformation protocols are not susceptible

for agrobacterium-mediated transformation. The mecha-

nism by which the BBM protein closes the gap in com-

petencies for transformation and regeneration in pepper is

not clear. BBM might exert a positive effect on the trans-

formation efficiency by creating a cellular environment that

is both susceptible to transformation and regeneration, or

by increasing the total number of regenerating cells (SLS),

thereby increasing the probability that both processes

coincide in one cell. 35S::BBM:GR explants not only pro-

duce a higher number of SLS than control explants; they

also elongate to produce shoots at a higher frequency.

Again, the underlying mechanism is not clear. Both

35S::GUS and 35S::BBM:GR explants initially produce

morphologically similar SLS that grow in dense rosettes

with no clear boundaries between them, suggesting a non-

functional or missing SAM. Somewhere during the

regeneration of BBM-SLS, a functional SAM and vascular

bundles are formed that allow the shoots to elongate and

develop into a normal plant. The ability of BBM to induce

direct regeneration, i.e., without an intervening callus

phase, may promote improved shoot polarity and

differentiation.

Efficient in vitro regeneration systems based on somatic

embryogenesis can facilitate the classical breeding process

by providing large amounts of clonal material for

Plant Cell Rep (2011) 30:1107–1115 1113

123

propagation, as well as explants for transformation. SE

production from immature zygotic embryos on solid

medium has been reported in C. annuum (chili and sweet

pepper types) and C. chinense although the induction fre-

quencies are low (maximum 8 SE/explant) (Binzel et al.

1996; Harini and Lakshmi Sita 1993; Steinitz et al. 2003),

and the SEs can exhibit a high frequency of morphological

defects that affects their conversion into seedlings (Steinitz

et al. 2003). Solıs-Ramos et al. (2009) used inducible

expression of the arabidopsis WUSCHEL (WUS) homeo-

box transcription factor (Zuo et al. 2002) to enhance SE

formation in C. chinense L. A small number of globular

structures could be induced to form on primary stem

explants transformed with the inducible WUS construct

butthe embryos failed to develop further and eventually

died. In contrast, stable 35S::BBM:GR transgenics exhib-

ited an extremely high regeneration capacity, producing

hundreds to thousands of well-formed embryos per explant

that could be converted at a high frequency into seedlings.

A number of strategies can be used to implement a

BBM-based transformation technology. For example, a

second gene of interest can be co-transformed along with

the 35S::BBM:GR construct or stable, highly regenerative

35S::BBM:GR transformants could be used as explants for

the introduction of a second gene of interest. In both

examples, the positive effect of the BBM protein on the

regeneration process can serve as a selectable marker

during (co-)transformation of a second gene of interest,

which can itself be selected in the classical way (e.g., using

antibiotic- or herbicide resistance) or via PCR. For some

purposes it may be desirable to avoid stable integration of

the 35S::BBM:GR transgene. This could be circumvented

by transient expression techniques (Vergunst et al. 2000) or

by segregation of the 35S::BBM:GR transgene in progeny

lines.

The sweet pepper transformation system described here

opens up possibilities for the introduction of new disease and

abiotic stress resistances, as well as for important repro-

ductive and architecture traits. In addition to these practical

applications, this transformation system provides opportu-

nities for building up fundamental research tools that can be

used to understand gene function in Capsicum spp.

Open Access This article is distributed under the terms of the

Creative Commons Attribution Noncommercial License which per-

mits any noncommercial use, distribution, and reproduction in any

medium, provided the original author(s) and source are credited.

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